Project Summary This proposal describes investigations in three main areas to understand how the organization of chromosomes in the nucleus, and their differentiation into heterochromatin and euchromatin, impacts chromosome breakage and repair, chromosome pairing, and chromosome replication in Drosophila melanogaster. In the first part, dicentric chromosomes are generated in the male germline, where they typically break, delivering a chromosome with a broken end to each daughter cell. The influence of structurally distinct centromeres and the amount of centromeric histone CenpA on the fate of these chromosomes will be examined. Experiments will be undertaken to understand how cells with a broken chromosome choose their fate, to repair or to die, and how they choose between different modes of repair. These choices have obvious relevance for human health. When a cell lives, but fails to repair damage, it may become cancerous. The mode of repair can determine whether gametes transmit a normal genome, or a genome with deficiencies or other structural variants or mutations. One particular mode of repair, Break Induced Replication, is known to be mutagenic in yeast, and has been implicated in chromosome change in humans. Chromosomes repaired by BIR will be examined to determine the types and frequencies of mutations produced in the germline of a higher eukaryote. In the second part of this work, the mechanism of mitotic chromosome pairing will be examined. Significant progress has been made in identifying genes that promote or inhibit pairing, but how homologous regions of chromosomes find each other to initiate pairing is still a mystery. This work will test various models for the initiation of pairing by studying the frequency of site-specific recombination within and between rearranged chromosomes. This work also has human health relevance, since failures of meiotic pairing can lead to gametes with chromosomal aneuploidy. Some cancer cells also show inappropriate homologous pairing, implying a possible connection between these chromosome and cellular states. In the third part of the proposed work, the timing of DNA replication in heterochromatin will be examined. Mutations in genes that encode proteins that affect heterochromatin will be tested to determine their effects on replication timing. Proper maintenance of heterochromatin is important for genome stability, and disruption of its normal pattern of replication can lead to altered gene expression, with impacts on cellular function and health.